This application addresses broad Challenge Area (15): Translational Science and specific Challenge Topic, 15- MH-103 Mapping the Neural Connectivity of a Mouse Model. Brain function is dictated by its circuitry, yet we know little about its wiring architecture: in the most-studied mammal (rat), only an estimated 10-30% of the long range circuit connections have been probed. The present Challenge Topic validates the growing consensus that it is time to close this gap by generating brainwide connectivity maps for model vertebrates. Over the last two years, we have organized several meetings involving the neuroanatomy community to gain in-depth understanding of the technical and scientific challenges of such a project. Based on this experience, we have designed and have begun to build and test an automated pipeline of experimental and computational techniques for achieving this goal. Our proposal is enabled by advances in automated wide-field slide scanning microscopy, decreasing data-storage costs, and established tract-tracing methods using injections of classical tracers and engineered viruses. The experimental plan can be summarized as follows. The mouse brain is divided into ~200 regions based on classical neuroanatomical and regional gene-expression data. For each region we inject one mouse with classical tracers and one mouse with viral tracers. From the injection site, the tracers are transported anterogradely to the area's projection targets and retrogradely to areas which project to the injection site. In this way, individual projections are revealed multiple times. In order to acquire this information, we will section the entire brain from each mouse and image the sections using an automated slide-scanning microscope. The resulting 2D slice-images will be combined in software to produce a 3D reconstructed brain image for each injection. Finally the 3D images from all of the individual injections will be combined by spatially registering them to the Allen Reference Atlas, ultimately generating a unified brainwide neural connectivity map. Generating the first unbiased, brainwide connectivity map in the mouse will have broad neuroscientific implications. The study of neural development, neural network modeling, evolutionary neuroanatomy, and associative and integrative brain function will benefit tremendously from finally having this landmark reference map to meaningfully constrain theories and aid in experimental design and interpretation of results. Relationships between gene expression and connectivity can be probed by analyzing the gene-expression maps generated by the Allen Institute in combination with the connectivity maps generated by this project. The baseline neural connectivity map generated in the present study will serve as a foundation for subsequently studying circuit polymorphisms across mutant mouse lines. The ability to objectively quantify alterations in connectivity in mouse models of neuropsychiatric disorders such as autism and schizophrenia will aid our understanding of their etiology and pathophysiology. Finally, our emphasis on open source software development, cost optimization and duplicability will result in an affordable, integrated instrument which other academic laboratories will be able to implement, so that this approach can be rapidly applied to a wide variety of neuroscientific problems. NARRATIVE The study of mouse models of neuropsychiatric disorders provides hope for the development of therapies for these burdensome illnesses, but progress has been slow due to the lack of knowledge about how the mouse brain is wired. This project aims to close this gap by generating the first brain-wide wiring diagram of mouse, automating techniques that are known to work but are labor-intensive. If successful, the project has the potential to fundamentally transform our understanding of the architecture of the normal and disordered brain.